[unreadable] [unreadable] Activation of the hedgehog (Hh) signaling pathway drives carcinogenesis in multiple organs, including skin, brain, skeletal muscle, pancreas, and prostate. Recent data indicate that the Hh pathway is also activated during breast carcinogenesis and that inhibition of Hh signaling by targeting the transmembrane receptor smoothened (Smo) reduces the growth of breast cancer cells. Small molecule Smo antagonists have been produced by several pharmaceutical companies and show promise as anti-cancer agents. In addition, cholecalciferol (vitamin D3) has also been demonstrated to bind Smo and inhibit its activity, suggesting a novel mechanism through which Vitamin D may work to prevent cancer. The purpose of this application is to provide new data in support of targeting Smo to prevent the progression of proliferative breast disease to invasive carcinoma and metastatic disease. The specific aims are to 1) test the effect of inhibition of Smo in vitro on the growth, migration, and invasion of breast epithelial cell lines representative of proliferative breast disease and breast cancer, 2) test the ability of the Smo antagonist cyclopamine to inhibit growth and progression of breast epithelial cells in vivo, and 3) assess expression of Smo, the Hh transcription factor Gli1and the ligand sonic hedgehog (Shh), as indicators of Hh signaling, in human breast carcinomas and correlate expression with overall survival. To accomplish these specific aims, cell lines representative of pre-invasive breast disease (MCF10AT and MCF10DCIS.com) and breast cancer will be treated with three different Smo antagonists - cyclopamine, KAAD-cyclopamine (a more potent analog of cyclopamine) and vitamin D3. The effect of each antagonist on clonogenic growth, invasion and migration will be assessed in vitro. Demonstration that the effect of the Smo antagonists is due to specific inhibition of Hh signaling will be accomplished by use of a Gli-luciferase reporter assay, assessment of expression of the Hh transcriptional target Gli1, and over-expression of a constitutively active form of Smo that is resistant to inhibition by Smo antagonists. A cell line that responds to Smo inhibition in vitro will be tested as xenografts in vivo for reductions in growth and progression after treatment with cyclopamine. Because Hh pathway activity is associated with advanced disease in other cancer types, a tissue microarray consisting of 218 breast cancers will be immunostained for expression of Smo, Gli1 and Shh and expression correlated with overall survival and pathologic indicators of prognosis. Smo and other members of the Hh pathway are new molecular targets that may prove effective in the prevention of breast cancer development and progression. This project will serve as an initial assessment of Smo antagonists as chemopreventive agents for breast cancer. [unreadable] [unreadable] [unreadable] [unreadable]